Enhanced immune response in bovine species

ABSTRACT

The present invention relates to a method of immune activation which is effective for eliciting a non-antigen-specific immune response in a member of the bovine species. The method is particularly effective for protecting a member of the bovine species from infectious disease and treating animals inflicted with infectious disease.

FIELD OF THE INVENTION

The present invention relates to a method of immune activation in amember of the bovine species. In particular, the present inventionincludes methods for eliciting systemic, non-specific andantigen-specific immune responses, which are useful for animaladministration and protection against infectious disease.

BACKGROUND OF THE INVENTION

Cattle are prime targets for many types of viral, bacterial, andparasite infections. Modern production practices, such as weaning,shipment of cattle, inclement weather, and nutritional needs within thebeef and dairy industries may also serve as risk factors that potentiatethe incidence of disease. Bovine respiratory disease (BRD), or bovinerespiratory diseases complex, as it is often referred to, occurs in bothdairy and beef cattle and is one of the leading causes of economic lossto the cattle industry throughout the world. These losses are due tomorbidity, mortality, reduced weight gains, treatment and preventioncosts, loss of milk production, and negative impacts on carcasscharacteristics.

The pathogenesis of BRD is thought to arise from numerous environmentaland physiological stressors, mentioned above, coupled with infectiousagents. Mannheimia (Pasteurella) haemolytica, Pasteurella multocida andHistophilus somni (formerly Haemophilus somnus) are considered part ofthe normal flora of the bovine upper-respiratory tract. Conversely, thelower respiratory tract is a relatively sterile environment that ismaintained by numerous immunological pathways aimed at the prevention ofmicrobial entry. When cattle are subjected to environmental andphysiological stressors, the animal's innate and acquired immunefunctions are compromised thereby allowing these aforementionedorganisms to proliferate and subsequently colonize the lower respiratorytract. Various bovine viruses are known to have immunosuppressiveeffects in the lung, such as infectious bovine rhinotracheitis virus(IBRV, IBR, or BHV 1), bovine viral diarrhea virus (BVDV), bovinerespiratory syncytial virus (BRSV), and parainfluenza type 3 virus(PI3). However, Mannheimia haemolytica is by far the most prevalentbacterial pathogen among cases of BRD.

Current prevention and treatment of BRD consists of antibioticadministration to populations of cattle upon arrival at feedlots (i.e.metaphylaxis), antibiotic therapy for sick cattle, and vaccinationagainst BRD viruses and bacteria including M. haemolytica.

There are different reasons why current vaccination programs andpharmaceutical therapies are not optimal to control BRD in cattle today.First, the host defense system plays a major role in combatinginfectious disease in cattle. Conventional treatments include theadministration of antibiotics to treat or control bacterial infections.However, there are no approved pharmaceutical treatments availableagainst viral infections. With BRD, in most cases not only is there abacterial infection but also a viral infection. Second, timing ofvaccination is often sub-optimal. For a respiratory vaccine to beoptimally effective the product should be administered 2-4 weeks priorto stress or shipment and this is typically not feasible in commercialcattle production. The vaccines are either administered too early or toolate to be optimally effective.

Therefore a need exists for a method to stimulate the immune system andbuild an offensive response to reduce or eliminate disease causingorganisms. It is important that this method is easy to administer, worksalone or in combination with vaccines or helps to make such vaccinesmore effective, has a longer duration or that does not require addedinjections to maximize immunity. The present invention provides a methodof eliciting a non-antigen-specific immune response in the bovinespecies that is easy to administer, works alone or in combination withvaccines, induces a protective response against one or more infectiousagents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1.1 graphically depicts average rectal temperature data accordingto dose of immunomodulator administered as described in Example 1.

FIG. 1.2 graphically depicts average daily weight gain data according todose of immunomodulator administered as described in Example 1.

FIG. 1.3 graphically depicts the model-adjusted lung lesion scores withrespect to dose of immunomodulator administered as described in Example1.

FIG. 2.1 graphically depicts average rectal temperature data accordingto dose of immunomodulator administered as described in Example 2.

FIG. 2.2 graphically depicts average daily weight gain data according todose of immunomodulator administered as described in Example 2.

FIG. 2.3 graphically depicts the model-adjusted lung lesion scores withrespect to dose of immunomodulator administered as described in Example2.

FIG. 3.1 graphically depicts the model-adjusted lung lesion scores withrespect to dose of immunomodulator administered as described in Example3

FIG. 3.2 graphically depicts the model-adjusted lung lesion scores withrespect to day of immunomodulator administration as described in Example3.

FIG. 4.1 graphically depicts % of protected animals by treatment groupas described in Example 4.

FIG. 4.2 graphically depicts percent of animals protected by treatmentgroup (<1% lung lesions and no lung lesions) as described in Example 4.

FIG. 5.1 graphically depicts measurements of the CD 25 EI expressionindex (y-axis) in cells infected with BHV-1 across all five cell typesfor each of the 6 treatment groups (x-axis) as described in Example 5.

FIG. 5.2 graphically depicts measurements of the CD 25 EI expressionindex (y-axis) in cells infected with BRSV across all five cell typesfor each of the 6 treatment groups (x-axis) as described in Example 5.

FIG. 5.3 graphically depicts measurements of the CD 25 EI expressionindex (y-axis) in cells infected with BVDV type 1 across all five celltypes for each of the 6 treatment groups (x-axis) as described inExample 5.

FIG. 5.4 graphically depicts measurements of the CD 25 EI expressionindex (y-axis) in cells infected with BVDV type 2 across all five celltypes for each of the 6 treatment groups (x-axis) as described inExample 5.

FIG. 5.5 graphically depicts measurements of the IFNγ expression index(y-axis) in cells infected with BHV-1 across all five cell types foreach of the 6 treatment groups (x-axis) as described in Example 5.

FIG. 5.6 graphically depicts measurements of the IFNγ expression index(y-axis) in cells infected with BRSV across all five cell types for eachof the 6 treatment groups (x-axis) as described in Example 5.

FIG. 5.7 graphically depicts measurements of the IFNγ expression index(y-axis) in cells infected with BVDV type 1 across all five cell typesfor each of the 6 treatment groups (x-axis) as described in Example 5.

FIG. 5.8 graphically depicts measurements of the IFNγ expression index(y-axis) in cells infected with BVDV type 2 across all five cell typesfor each of the 6 treatment groups (x-axis) as described in Example 5.

FIG. 5.9 graphically depicts measurements of the IL-4 expression index(y-axis) in cells infected with BHV-1 across all five cell types foreach of the 6 treatment groups (x-axis) as described in Example 5.

FIG. 5.10 graphically depicts measurements of the IL-4 expression index(y-axis) in cells infected with BRSV across all five cell types for eachof the 6 treatment groups (x-axis) as described in Example 5.

FIG. 5.11 graphically depicts measurements of the IL-4 expression index(y-axis) in cells infected with BVDV type 1 across all five cell typesfor each of the 6 treatment groups (x-axis) as described in Example 5.

FIG. 5.12 graphically depicts measurements of the IL-4 expression index(y-axis) in cells infected with BVDV type 2 across all five cell typesfor each of the 6 treatment groups (x-axis) as described in Example 5.

FIG. 5.13 graphically depicts Model adjusted serum antibody titerestimates (y-axis) in cells infected with BVDV type 1 across all fivecell types for each of the 6 treatment groups (x-axis) as described inExample 5.

FIG. 5.14 graphically depicts Model adjusted serum antibody titerestimates (y-axis) in cells infected with BVDV type 2 across all fivecell types for each of the 6 treatment groups (x-axis) as described inExample 5.

FIG. 5.15 graphically depicts Model adjusted serum antibody titerestimates (y-axis) in cells infected with BHV-1 across all five celltypes for each of the 6 treatment groups (x-axis) as described inExample 5.

FIG. 5.16 graphically depicts model-adjusted average daily gain outcomesas described in Example 5.

FIG. 6.1 graphically depicts the BHV1 SNT titers for the treatmentgroups as described in Example 6.

DETAILED DESCRIPTION OF THE INVENTION

The method of eliciting an immune response in a member of the bovinespecies of the present invention includes administering to the member ofthe bovine species an effective amount of an immunomodulator compositionto elicit an immune response. The immunomodulator composition includes aliposome delivery vehicle and at least one nucleic acid molecule. Inaddition, the immunomodulator elicits a non-antigen-specific immuneresponse that is effective alone or enhances the operation of at leastone biological agent such as a vaccine, when administered prior to sucha vaccine, co-administered with such a vaccine, administered postvaccination, or mixed with the vaccine.

The methods provide new treatment strategies for protecting the bovinespecies from infectious diseases and treating populations havinginfectious disease. Finally, the method of the present inventionprovides a more rapid, a longer and better protection against a diseasewhen the immunomodulator is used in combination with a vaccine.

1. Composition

a. Immunomodulator

In one embodiment of the invention, the immunomodulator compositionincludes a liposome delivery vehicle and at least one nucleic acidmolecule, as described in U.S. Pat. No. 6,693,086, and incorporatedherein by reference.

A suitable liposome delivery vehicle comprises a lipid composition thatis capable of delivering nucleic acid molecules to the tissues of thetreated subject. A liposome delivery vehicle is preferably capable ofremaining stable in a subject for a sufficient amount of time to delivera nucleic acid molecule and/oro biological agent. In one embodiment, theliposome delivery vehicle is stable in the recipient subject for atleast about 5 minutes. In another embodiment, the liposome deliveryvehicle is stable in the recipient subject for at least about 1 hour. Inyet another embodiment, the liposome delivery vehicle is stable in therecipient subject for at least about 24 hours.

A liposome delivery vehicle of the present invention comprises a lipidcomposition that is capable of fusing with the plasma membrane of a cellto deliver a nucleic acid molecule into a cell. In one embodiment, whendelivered a nucleic acid: liposome complex of the present invention isat least about 1 picogram (pg) of protein expressed per milligram (mg)of total tissue protein per microgram (μg) of nucleic acid delivered. Inanother embodiment, the transfection efficiency of a nucleic acid:liposome complex is at least about 10 pg of protein expressed per mg oftotal tissue protein per μg of nucleic acid delivered; and in yetanother embodiment, at least about 50 pg of protein expressed per mg oftotal tissue protein per μg of nucleic acid delivered. The transfectionefficiency of the complex may be as low as 1 femtogram (fg) of proteinexpressed per mg of total tissue protein per μg of nucleic aciddelivered, with the above amounts being more preferred.

A preferred liposome delivery vehicle of the present invention isbetween about 100 and 500 nanometers (nm), in another embodiment,between about 150 and 450 nm and in yet another embodiment, betweenabout 200 and 400 nm in diameter.

Suitable liposomes include any liposome, such as those commonly used in,for example, gene delivery methods known to those of skill in the art.Preferred liposome delivery vehicles comprise multilamellar vesicle(MLV) lipids and extruded lipids. Methods for preparation of MLV's arewell known in the art. More preferred liposome delivery vehiclescomprise liposomes having a polycationic lipid composition (i.e.,cationic liposomes) and/or liposomes having a cholesterol backboneconjugated to polyethylene glycol. Exemplary cationic liposomecompositions include, but are not limited to.N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)and cholesterol, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniumchloride (DOTAP) and cholesterol,1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride(DOTIM) and cholesterol, dimethyldioctadecylammonium bromide (DDAB) andcholesterol, and combinations thereof. A most preferred liposomecomposition for use as a delivery vehicle includes DOTIM andcholesterol.

A suitable nucleic acid molecule includes any nucleic acid sequence suchas coding or non-coding sequence, and DNA or RNA. Coding nucleic acidsequences encode at least a portion of a protein or peptide, whilenon-coding sequence does not encode any portion of a protein or peptide.According to the present invention, “non-coding” nucleic acids caninclude regulatory regions of a transcription unit, such as a promoterregion. The term, “empty vector” can be used interchangeably with theterm “non-coding”, and particularly refers to a nucleic acid sequence inthe absence of a protein coding portion, such as a plasmid vectorwithout a gene insert. Expression of a protein encoded by the nucleicacid molecule is not required for elicitation of a non-antigen-specificimmune response; therefore the nucleic acid molecule does notnecessarily need to be operatively linked to a transcription controlsequence. However, further advantages may be obtained (i.e.,antigen-specific and enhanced immunity) by including in the compositionnucleic acid sequence (DNA or RNA) which encodes an immunogen and/or acytokine.

Complexing a liposome with a nucleic acid molecule may be achieved usingmethods standard in the art or as described in U.S. Pat. No. 6,693,086,and incorporated herein by reference. A suitable concentration of anucleic acid molecule to add to a liposome includes a concentrationeffective for delivering a sufficient amount of nucleic acid moleculeinto a subject such that a systemic immune response is elicited. In oneembodiment, from about 0.1 μg to about 10 μg of nucleic acid molecule iscombined with about 8 nmol liposomes, in another embodiment, from about0.5 μg to about 5 μg of nucleic acid molecule is combined with about 8nmol liposomes, and in yet another embodiment, about 1.0 μg of nucleicacid molecule is combined with about 8 nmol liposomes. In oneembodiment, the ratio of nucleic acids to lipids (pg nucleic acid: nmollipids) in a composition is at least about 1:1 nucleic acid: lipid byweight (i.e., 1 μg nucleic acid: 1 nmol lipid), and in anotherembodiment, at least about 1:5, and in yet another embodiment, at leastabout 1:10, and in a further embodiment at least about 1:20. Ratiosexpressed herein are based on the amount of cationic lipid in thecomposition, and not on the total amount of lipid in the composition. Inanother embodiment, the ratio of nucleic acids to lipids in acomposition of the invention is from about 1:1 to about 1:80 nucleicacid: lipid by weight; and in another embodiment, from about 1:2 toabout 1:40 nucleic acid: lipid by weight; and a further embodiment, fromabout 1:3 to about 1:30 nucleic acid: lipid by weight; and in yetanother embodiment, from about 1:6 to about 1:15 nucleic acid: lipid byweight.

b. Biological Agent

In another embodiment of the invention, the immunomodulator includes aliposome delivery vehicle, a nucleic acid molecule, and at least onebiological agent.

Suitable biological agents are agents that are effective in preventingor treating bovine disease. Such biological agents include immuneenhancer proteins, immunogens, vaccines, antimicrobials or anycombination thereof. Suitable immune enhancer proteins are thoseproteins known to enhance immunity. By way of a non-limiting example, acytokine, which includes a family of proteins, is a known immunityenhancing protein family. Suitable immunogens are proteins which elicita humoral and/or cellular immune response such that administration ofthe immunogen to a subject mounts an immunogen-specific immune responseagainst the same or similar proteins that are encountered within thetissues of the subject. An immunogen may include a pathogenic antigenexpressed by a bacterium, a virus, a parasite or a fungus. Preferredantigens include antigens which cause an infectious disease in asubject. According to the present invention, an immunogen may be anyportion of a protein, naturally occurring or synthetically derived,which elicits a humoral and/or cellular immune response. As such, thesize of an antigen or immunogen may be as small as about 5-12 aminoacids and as large as a full length protein, including sizes in between.The antigen may be a multimer protein or fusion protein. The antigen maybe purified peptide antigens derived from native or recombinant cells.The nucleic acid sequences of immune enhancer proteins and immunogensare operatively linked to a transcription control sequence, such thatthe immunogen is expressed in a tissue of a subject, thereby elicitingan immunogen-specific immune response in the subject, in addition to thenon-specific immune response.

In another embodiment of the invention, the biological agent is avaccine. The vaccine may include a live, infectious, viral, bacterial,or parasite vaccine or a killed, inactivated, viral, bacterial, orparasite vaccine. In one embodiment, one or more vaccines, live orkilled viral vaccines, may be used in combination with theimmunomodulator composition of the present invention. Suitable vaccinesinclude those known in the art for the cattle species. Exemplaryvaccines, without limitation, include those used in the art forprotection against infectious bovine rhinotracheitis (IBR) (Type 1bovine herpes virus (BHV1)), parainfluenza virus type 3 (PI3), bovinerespiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDVType 1 and 2). Histophilus somni, Mycoplasma bovis, and other diseasesknown in the art. In an exemplary embodiment, a vaccine for theprotection against Mannheimia haemolytica may be used in combinationwith the immunomodulator composition of the present invention.

In yet another embodiment of the invention, the biological agent is anantimicrobial. Suitable antimicrobials include: quinolones, preferablyfluoroquinolones, β-lactams, and macrolide-streptogramin-lincosamide(MLS) antibiotics.

Suitable quinolones include benofloxacin, binfloxacin, cinoxacin,ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin,enrofloxacin, fleroxacin, gemifloxacin, ibafloxacin, levofloxacin,lomefloxacin, marbofloxacin, moxifloxacin, norfloxacin, ofloxacin,orbifloxacin, pazufloxacin, pradofloxacin, perfloxacin, temafloxacin,tosufloxacin, sarafloxacin, gemifloxacin, and sparfloxacin. Preferredfluoroquinolones include ciprofloxacin, enrofloxacin, moxifloxacin,danofloxacin, and pradofloxacin. Suitable naphthyridones includenalidixic acid.

Suitable β-lactams include penicillins, such as benzathine penicillin,benzylpenicillin (penicillin G), phenoxymethylpenicillin (penicillin V),procaine penicillin, methicillin, oxacillin, nafcillin, cloxacillin,dicloxacillin, flucloxacillin, temocillin, amoxicillin, ampicillin,co-amoxiclav (amoxicillin and clavulanic acid), azlocillin,carbenicillin, ticarcillin, mezlocillin, piperacillin; cephalosporins,such as cefalonium, cephalexin, cefazolin, cefapririn, cefquinome,ceftiofur, cephalothin, cefaclor, cefuroxime, cefamandole, defotetan,cefoxitin, ceftriaxone, cefotaxime, cefpodoxime, cefixime, ceftazidime,cefepime, cefpirome; carbapenems and penems such as imipenem, meropenem,ertapenem, faropenem, doripenem, monobactams such as aztreonam(Azactam), tigemonam, nocardicin A, tabtoxinine-B-lactam; andβ-lactamase inhibitors such as clavulanic acid, tazobactam, andsulbactam. Preferred β-lactams include cephalosporins, in particular,cefazolin.

Suitable MLS antibiotics include any macrolide, lincomycin, clindamycin,pirlimycin. A preferred lincosamide is pirlimycin.

Other antimicrobials include 2-pyridones, tetracyclines, sulfonamides,aminoglycosids, trimethoprim, dimetridazoles, erythromycin, framycetin,furazolidone, various pleuromutilins such as tiamulin, valnemulin,various, streptomycin, clopidol, salinomycin, monensin, halofuginone,narasin, robenidine, etc.

2. Methods

a. Methods of Immune Stimulation

In one embodiment of the invention, an immune response is elicited in amember of the bovine species by administering an effective amount of animmunomodulator composition to the member of the bovine species. Theeffective amount is sufficient to elicit an immune response in themember of the bovine species. The immunomodulator includes a liposomedelivery vehicle and a nucleic acid molecule.

In one embodiment, the effective amount of the immunomodulator is fromabout 1 micrograms to about 1000 micrograms per animal. In anotherembodiment, the effective amount of the immunomodulator is from about 5micrograms to about 500 micrograms per animal. In yet anotherembodiment, the effective amount of the immunomodulator is from about 10micrograms to about 100 micrograms per animal. In a further embodiment,the effective amount of the immunomodulator is from about 10 microgramsto about 50 micrograms per animal.

In another embodiment of the invention, an immune response is elicitedin a member of the bovine species by administering an effective amountof an immunomodulator, which includes a liposome delivery vehicle, anisolated nucleic acid molecule, and a biological agent. It iscontemplated that the biological agent may be mixed with orco-administered with the immunomodulator or independently thereof.Independent administration may be prior to or after administration ofthe immunomodulator. It is also contemplated that more than oneadministration of the immunomodulator or biological agent may be used toextend enhanced immunity. Furthermore, more than one biological agentmay be co-administered with the immunomodulator, administered prior tothe immunomodulator, administered after administration of theimmunomodulator, or concurrently.

b. Diseases

The methods of the invention elicit an immune response in a subject suchthat the subject is protected from a disease that is amenable toelicitation of an immune response. As used herein, the phrase “protectedfrom a disease” refers to reducing the symptoms of the disease; reducingthe occurrence of the disease, and reducing the clinical or pathologicseverity of the disease or reducing shedding of a pathogen causing adisease. Protecting a subject can refer to the ability of a therapeuticcomposition of the present invention, when administered to a subject, toprevent a disease from occurring, cure, and/or alleviate or reducedisease symptoms, clinical signs, pathology, or causes. Examples ofclinical signs of BRD include lung lesions, increased temperature,depression (e.g. anorexia, reduced responsiveness to external stimuli,droopy ears), nasal discharge, and respiratory character (e.g.respiratory rate, respiratory effort). As such, to protect a member ofthe bovine species from a disease includes both preventing diseaseoccurrence (prophylactic treatment) and treating a member of the bovinespecies that has a disease (therapeutic treatment). In particular,protecting a subject from a disease is accomplished by eliciting animmune response in the member of the bovine species by inducing abeneficial or protective immune response which may, in some instances,additionally suppress, reduce, inhibit, or block an overactive orharmful immune response. The term “disease” refers to any deviation fromthe normal health of a member of the bovine species and includes a statewhen disease symptoms are present, as well as conditions in which adeviation (e.g., infection, gene mutation, genetic defect, etc.) hasoccurred, but symptoms are not yet manifested.

Methods of the invention may be used for the prevention of disease,stimulation of effector cell immunity against disease, elimination ofdisease, alleviation of disease, and prevention of a secondary diseaseresulting from the occurrence of a primary disease.

The present invention may also improve the acquired immune response ofthe animal when co-administered with a vaccine versus administration ofthe vaccine by itself. Generally a vaccine once administered does notimmediately protect the animal as it takes time to stimulate acquiredimmunity. The term “improve” refers, in the present invention, toelicitation of an innate immune response in the animal until the vaccinestarts to protect the animal and/or to prolong the period of protection,via acquired immunity, given by the vaccine.

Methods of the invention include administering the composition toprotect against infection of a wide variety of pathogens. Thecomposition administered may or may not include a specific antigen toelicit a specific response. It is contemplated that the methods of theinvention will protect the recipient subject from disease resulting frominfectious microbial agents including, without limitation, viruses,bacteria, fungi, and parasites. Exemplary viral infectious diseases,without limitation, include those resulting from infection withinfectious bovine rhinotracheitis (IBR) (Type 1 bovine herpes virus(BHV1)), parainfluenza virus type 3 (PI3), bovine respiratory syncytialvirus (BRSV), bovine viral diarrhea virus (BVDV Type 1 and 2), bovineadenovirus, bovine coronavirus (BCV), bovine calicivirus, bovineparvovirus, BHV4, bovine reovirus, bovine enterovirus, bovinerhinovirus, malignant catarrhal fever virus, bovine leukemia virus,rabies virus, Vesicular stomatitis virus (VSV), bluetongue (Orbivirus),recombinants thereof, and other viruses known in the art. Exemplarybacterial infections, without limitation, include those resulting frominfection with gram positive or negative bacteria and Mycobacteriasuchas Escherichia coli, Pasteurella multocida, Clostridium perfringens,Clostridium colinum, Campylobacter jejuni, Clostridium botulinum,Clostridium novyi, Clostridium chauveoi, Clostridium septicum,Clostridium hemolyticum, Clostridium tetani, Mannheimia haemolytica,Ureaplasma diversum, Mycoplasma dispar, Mycoplasma bovis, Mycoplasmabovirhinis, Histophilus somni, Campylobacter fetus, Leptospira spp.,Arcanobacterium pyogenes, Bacillus anthrax, Fusobacterium necrophorum,Fusobacterium spp., Treponema spp., Corynebacterium, Brucella abortus,Mycobacterium paratuberculosis, Mycobacterium spp., Histophilus spp.,Moraxella spp., Muellerius spp., Mycoplasma spp., Salmonella spp.,Bacillus anthracis, and other bacteria known in the art. Exemplary fungior mold infection, without limitation, include those resulting frominfection with Actinobacterim spp., Aspergillus spp., and Histomonasspp., and other infectious fungi or mold known in the art. Exemplaryparasites include, without limitation. Neospora spp., Trichostrongylus,Cooperia, Anaplasma spp, Babesia spp, Chorioptes spp, Cysticercus spp,Dermatophilus spp, Damalinia bovis, Dictylocaulus spp, Eimeria spp,Eperythrozoon spp, Haemonchus spp, Melophagus spp, Muellerius spp,Nematodirus spp, Oestrus spp, Ostertagia spp, Psoroptes spp, Sarcoptesspp, Serpens spp, Strongyloides spp, Toxoplasma spp, Trichuris spp,Trichophyton spp, and Tritrichomas spp, Fascioloides spp, Anaplasmamarginale, and other parasites known in the art.

c. Subjects

The methods of the invention may be administered to any subject ormember of the bovine species, whether domestic or wild. In particular,it may be administered to those subjects that are commercially rearedfor breeding, meat or milk production. Suitable bovine subjects, withoutlimitation, include antelopes, buffalos, yaks, cattle, and bison. In oneembodiment, the member of the bovine species is cattle. Species ofcattle include, without limitation, cows, bulls, steers, heifer, ox,beef cattle, or dairy cattle. A skilled artisan will appreciate that themethods of the invention will be largely beneficial to cattle reared forbreeding, meat or milk production, since they are especially vulnerableto environmental exposure to infectious agents.

d. Administration

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular biological agentsselected, the age and general health status of the subject, theparticular condition being treated and the dosage required fortherapeutic efficacy. The methods of this invention may be practicedusing any mode of administration that produces effective levels of animmune response without causing clinically unacceptable adverse effects.The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art.

Vaccination of the bovine species can be performed at any age. Thevaccine may be administered intravenously, intramuscularly, intradermal,intraperitoneal, subcutaneously, by spray/aerosol, orally,intraocularly, intratracheally, intranasal, or by other methods known inthe art. Further, it is contemplated that the methods of the inventionmay be used based on routine vaccination schedules. The immunomodulatormay also be administered intravenously, intramuscularly, subcutaneously,by spray, orally, intraocularly, intratracheally, nasally, or by othermethods known in the art. In one embodiment, the immunomodulator isadministered subcutaneously. In another embodiment, the immunomodulatoris administered intramuscularly. In yet another embodiment, theimmunomodulator is administered as a spray. In a further embodiment, theimmunomodulator is administered orally.

In one embodiment, the immunomodulator is administered by itself to theanimal prior to challenge (or infection). In another embodiment, theimmunomodulator is administered by itself to the animal post challenge(or infection). In yet another embodiment, the immunomodulator isadministered by itself to the animal at the same time as challenge (orinfection). In a further embodiment, the immunomodulator composition isco-administered at the same time as the vaccination prior to challenge.In yet a further embodiment, the immunomodulator composition isco-administered at the same time as the vaccination at the same time aschallenge (or infection). The co-administration may includeadministering the vaccine and immunomodulator in the same generallocation on the animal at two different sites next to each other (i.e.,injections next to each other at the neck of the animal), on opposingsides of the animal at the same general location (i.e., one on each sideof the neck), or on different locations of the same animal. In anotherembodiment, the immunomodulator composition is administered prior tovaccination and challenge. In a further embodiment, the immunomodulatorcomposition is administered after vaccination but prior to challenge. Ina further embodiment, the immunomodulator composition is administeredafter challenge to an animal that has been vaccinated prior to challenge(or infection).

In one embodiment, the immunomodulator is administered from about 1 toabout 14 days prior to challenge or from about 1 to about 14 days postchallenge. In another embodiment, the immunomodulator is administeredfrom about 1 to about 7 days prior to challenge or from about 1 to about7 days post challenge. In yet another embodiment, the immunomodulator isadministered 1, 2, 3, 4, 5, 6, 7 days prior to challenge or 1, 2, 3, 4,5, 6, 7 days post challenge.

Other delivery systems may include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compositions therefore increasing convenience.Many types of release delivery systems are available and known to thoseof ordinary skill in the art. They include polymer based systems such aspoly(lactide-glycolide), copolyoxalates, polycaprolactones,polyesteramides, polyorthoesters, polyhydroxybutyric acid, andpolyanhydrides. Microcapsules of the foregoing polymers containing drugsare described in, for example, U.S. Pat. No. 5,075,109. Delivery systemsalso include non-polymer systems that are lipids including sterols suchas cholesterol, cholesterol esters and fatty acids or neutral fats suchas mono-di and tri-glycerides; hydrogel release systems; sylasticsystems; peptide based systems; wax coatings; compressed tablets usingconvention binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to erosionalsystems in which an agent of the invention is contained in a form withina matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,675,189and 5,736,152, and diffusional systems in which an active componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-basedhardware delivery systems can be used, some of which are adapted forimplantation.

As various changes could be made in the above composition, products andmethods without departing from the scope of the invention, it isintended that all matter contained in the above description and in theexamples given below, shall be interpreted as illustrative and not in alimiting sense.

DEFINITIONS

The term “effective amount” refers to the amount necessary or sufficientto realize a desired biologic effect. For example, an effective amountof immunomodulator for treating or preventing an infectious disease isthat amount necessary to cause the development of an immune responseupon exposure to the microbe, thus causing a reduction in the amount ofmicrobe within the subject and preferably to the eradication of themicrobe. The effective amount for any particular application can varydepending on such factors as the disease or condition being treated, thesize of the subject, or the severity of the disease or condition. One ofordinary skill in the art can empirically determine the effective amountof immunomodulator without necessitating undue experimentation.

The term “cytokine” refers to an immune enhancing protein family. Thecytokine family includes hematopoietic growth factor, interleukins,interferons, immunoglobulin superfamily molecules, tumor necrosis factorfamily molecules and chemokines (i.e. proteins that regulate themigration and activation of cells, particularly phagocytic cells).Exemplary cytokines include, without limitation, interleukin-2 (IL-2),interleukin-12 (IL12), interleukin-15 (IL-15), interleukin-18 (IL-18),interferon-α (IFNα), and interferon-γ (IFNγ).

The term “elicit” can be used interchangeably with the terms activate,stimulate, generate or upregulate.

The term “eliciting an immune response” in a subject refers tospecifically controlling or influencing the activity of the immuneresponse, and can include activating an immune response, upregulating animmune response, enhancing an immune response and/or altering an immuneresponse (such as by eliciting a type of immune response which in turnchanges the prevalent type of immune response in a subject from onewhich is harmful or ineffective to one which is beneficial orprotective).

The term “operatively linked” refers to linking a nucleic acid moleculeto a transcription control sequence in a manner such that the moleculeis able to be expressed when transfected (i.e., transformed, transducedor transfected) into a host cell. Transcriptional control sequences aresequences which control the initiation, elongation, and termination oftranscription. Particularly important transcription control sequencesare those which control transcription initiation, such as promoter,enhancer, operator and repressor sequences. A variety of suchtranscription control sequences are known to those skilled in the art.Preferred transcription control sequences include those which functionin avian, fish, mammalian, bacteria, plant, and insect cells. While anytranscriptional control sequences may be used with the invention, thesequences may include naturally occurring transcription controlsequences naturally associated with a sequence encoding an immunogen orimmune stimulating protein.

The terms “nucleic acid molecule” and “nucleic acid sequence” can beused interchangeably and include DNA. RNA, or derivatives of either DNAor RNA. The terms also include oligonucleotides and larger sequences,including both nucleic acid molecules that encode a protein or afragment thereof, and nucleic acid molecules that comprise regulatoryregions, introns, or other non-coding DNA or RNA. Typically, anoligonucleotide has a nucleic acid sequence from about 1 to about 500nucleotides, and more typically, is at least about 5 nucleotides inlength. The nucleic acid molecule can be derived from any source,including mammalian, fish, bacterial, insect, viral, plant, or syntheticsources. A nucleic acid molecule can be produced by methods commonlyknown in the art such as recombinant DNA technology (e.g., polymerasechain reaction (PCR), amplification, cloning) or chemical synthesis.Nucleic acid molecules include natural nucleic acid molecules andhomologues thereof, including, but not limited to, natural allelicvariants and modified nucleic acid molecules in which nucleotides havebeen inserted, deleted, substituted, or inverted in such a manner thatsuch modifications do not substantially interfere with the nucleic acidmolecule's ability to encode an immunogen or immune stimulating proteinuseful in the methods of the present invention. A nucleic acid homologuemay be produced using a number of methods known to those skilled in theart (see, for example. Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Labs Press, 1989), which is incorporatedherein by reference. Techniques to screen for immunogenicity, such aspathogen antigen immunogenicity or cytokine activity are known to thoseof skill in the art and include a variety of in vitro and in vivoassays.

EXAMPLES

The following examples illustrate various embodiments of the invention.

Example 1 Evaluation of Cattle Receiving a DNA Immunomodulator Before orAfter Developing Natural Bovine Respiratory Disease

The purpose of this study was to determine the efficacy of the DNAimmunomodulator administered to calves prior to and after developingnatural cases of BRD.

Immunomodulator

The immunomodulator used in this study was a composition comprising acationic lipid and non-coding DNA. The synthetic immunomodulator lipidcomponents[1-[2-[9-(Z)-octadecenoyloxy]]-2-[8](Z)-heptadecenyl]-3-[hydroxyethyl]imidazoliniumchloride (DOTIM) and a synthetic neutral lipid cholesterol wereformulated to produce liposomes approximately 200 nm in diameter (See,U.S. Pat. No. 6,693,086). The DNA component was a 4242 base-pairnon-coding DNA plasmid produced in E. coli, which, being negativelycharged, associates with the positively-charged (cationic) liposomes(See, U.S. Pat. No. 6,693,086).

Study Animals

84 Holstein steer calves of weaning age were selected from a herdwithout a current history of respiratory disease. Each individual calfwas initially evaluated and determined to be in good health. The 84calves were divided into seven treatment groups of 12 calves each. Onlyanimals not vaccinated for Mannheimia haemolytica were included in thestudy. None of the animals had received an antimicrobial agent within 30days prior to administration of DNA immunomodulator.

The treatment groups were administered varying doses of the DNAimmunomodulator describe above on the day of treatment as indicated inTable 1.1 below. The dilution scheme of the DNA immunomodulator isprovided in Table 1.2. The DNA immunomodulator was administeredintramuscularly and cranial to the left shoulder, ventral to the nuchalligament, and caudo-dorsal to the jugular groove of the calves.

As referred to below, Treatment Day −1 refers to the start date of thestudy after initial selection in which the calves were evaluated anddetermined to be suitable for the study. Treatment Day 0 is one daysubsequent to Day −1, and so on.

TABLE 1.1 Administration Schedule of Immunomodulator DNA Day of AnimalsTreat- Immuno- Immuno- per ment modulator modulator Treatment numberDose (μg) Administration group 1 500 −1 12 2 200 −1 12 3 50 −1 12 4 5000 12 5 200 0 12 6 50 0 12 7 0 (Control) NA 12

A large proportion of the calves were observed to be experiencingvariable levels of BRD on the morning of Day 0. By Day 5 all of thecalves remaining in the study population were observed to have met thecase definition for BRD morbidity. Cattle were only removed from thestudy population if euthanasia was indicated due to severe BRD. No otherinfectious/non-infectious diseases were observed and thereby requiredremoval in this study.

Evaluation

On Days 1-5 of the study the calves were evaluated for various healthindicators. For example, rectal temperature and average daily weightwere determined for each of the calves per day through the length of thestudy. Animals were evaluated at approximately the same time each day(+/−3 hours) from Day 1 to Day 5. FIGS. 1.1 and 1.2 present the averagesof rectal temperatures and average daily weight gain according to doseof immunomodulator administered.

On Day 5, all calves were euthanized and necropsied. Lung lesion scoreswere determined (based upon the degree lung consolidation estimated byvisual inspection and manual palpation) for each individual calf at thetime of necropsy.

FIG. 1.3 presents the lung lesion scores with respect to dose ofimmunomodulator administered. The overall lung lesion scores for eachday of administration were approximately 11% and 14% for Day −1 and Day0, respectively. Lung lesion scores of 11.2%, 9.0%, 10.8% and 19.9% wereexhibited for 500, 200, 50 and negative control groups, respectively.The largest difference between the control group and a treated group(200 μg) was about an 11% reduction.

Model-adjusted estimates on FIG. 1.3 reflect the raw averages that areadjusted for all statistical model covariates (i.e. dose, day, anddose×day) as well as for the pen in which the calves were housedthroughout the study. Therefore, model-adjusted estimates may displaydifferences compared to the raw averages.

Subsequent bacteriology (lung cultures) and virology (nasal swabs) werealso performed. Of the remaining calves (69) that were euthanized on Day5, 11.6% were found to be shedding bovine herpes virus type 1 (BHV-1) innasal secretions. With regard to lung cultures from all of the studyanimals, 41% were positive for Mh, 31.3% were culture positive forPasteurella multocida (Pm), 10.8% were culture positive for both Mh andPm, and no Histophilus somni was isolated throughout the studypopulation. Cultures for Mycoplasma bovis were not performed in thisstudy

Results

In this study, the dose of the DNA immunomodulator (i.e. 500 μg, 200 μg,and 50 μg) approached a significant reduction in lung lesion scorescompared to the negative control (P=0.1284; See FIG. 1.3). However, theday of DNA immunomodulator administration (i.e., Day −1 or 0) was notsignificantly associated with lung lesion scores. No statisticaldifferences in lung lesion scores were observed among the DNAimmunomodulator dose groups. Rectal temperature tended to besignificantly associated with the dose of DNA immunomodulator (P=0.1190)but was not associated with the day of administration. No obviousdifferences were observed between the dose of the DNA immunomodulatorand the negative control with regard to average daily weight gain.

There was a strong tendency for the DNA immunomodulator to reduce lunglesions compared to negative control, thereby, providing evidence thatthis product has the potential to protect lung tissue during a BRDoutbreak. In this study, the day of treatment administration was notassociated with lung lesions thereby indicating that it does not matterif cattle received the DNA immunomodulator one day prior or the same dayas the onset of clinical signs associated with BRD. This outcome isimportant as the timing of exposure to BRD pathogens is generallyunknown among typical production systems and is further complicated bythe impact of various stressors experienced by cattle throughout thechain of production. Therefore, providing producers with a product thatoffers flexibility in the timing of administration, in relation to theonset of BRD, is of extreme value in the beef and dairy industries.

Example 2 Evaluation of Cattle Receiving a DNA ImmunomodulatorConcurrently with or One Day After an Experimental Challenge withMannheimia haemolytica

The purpose of this study was to determine the efficacy of the DNAimmunomodulator administered to calves concurrently with or one dayafter an experimental challenge with Mannheimia haemolytica.

Immunomodulator

The immunomodulator used in this study was the composition describedabove in Example 1.

Study Animals

84 Holstein steer calves of weaning age and weighing on average about300 lbs (136 kg) were selected from a herd without a current history ofrespiratory disease. Each individual calf was initially evaluated anddetermined to be in good health. The 84 calves were divided into seventreatment groups of 12 calves each. Only animals not vaccinated forMannheimia haemolytica were included in the study. None of the animalshad received an antimicrobial agent within 30 days prior toadministration of DNA immunomodulator. The treatment groups wereadministered varying doses of the DNA immunomodulator on the day oftreatment as indicated in Table 2.1 below. The dilution scheme of theDNA immunomodulator is provided in Table 2.2. The DNA immunomodulatorwas administered intramuscularly and cranial to the left shoulder,ventral to the nuchal ligament, and caudo-dorsal to the jugular grooveof the calves.

As referred to below, Treatment Day 0 refers to the start date of thestudy after initial selection in which the calves were evaluated anddetermined to be in good health. Treatment Day 1 is one day subsequentto Day 0, and so on.

TABLE 2.1 Administration Schedule of Immunomodulator and Mh ChallengeDNA Day of Day of Mh Animals Treat- Immuno- Immuno- Challenge per mentmodulator modulator Adminis- Treatment number Dose (μg) Administrationtration group 1 500 0 0 12 2 200 0 0 12 3 50 0 0 12 4 500 1 0 12 5 200 10 12 6 50 1 0 12 7 0 (Control) NA 0 12

Experimental Challenge

On Day 0, the calves were challenged a total of 3.12×10⁷ colony formingunits (CFU) of Mannheimia haemolytica. The inoculum was administered viathe respiratory tract. By Day 3, all of the calves in the studypopulation were observed to have met the case definition for BRDmorbidity. The median day of onset was one day.

Evaluation

As in the previous example, on Days 1-5 of the study the calves wereevaluated for various health indicators. Rectal temperature and averagedaily weight were determined for each of the calves per day through thelength of the study. Animals were evaluated at approximately the sametime each day. FIGS. 2.1 and 2.2 present the averages of rectaltemperatures and average daily weight gains with respect to dose ofimmunomodulator administered.

On Day 5, all calves were euthanized and necropsied. Lung lesion scoreswere determined for each individual calf at the time of necropsyaccording to the formula described in Example 1.

FIG. 2.3 presents the model-adjusted lung lesion scores with respect todose of immunomodulator administered.

Results

In this study, the dose of the DNA immunomodulator (i.e. 500 μg, 200 μg,and 50 μg) significantly reduced lung lesion scores compared to thenegative control. However, the lower doses (200 μg, and 50 μg)outperformed the 500 μg dose in reducing lung lesions. The day of DNAimmunomodulator administration (i.e., Day 0 or 1) was not significantlyassociated with lung lesion scores. No statistical differences in lunglesion scores were observed among the DNA immunomodulator dose groups.Rectal temperature was significantly reduced in calves administered theDNA immunomodulator compared to the negative control, but was notassociated with dose. No obvious differences were observed between thedose of the DNA immunomodulator and the negative control with regard toaverage daily weight gain.

There was a strong tendency for the DNA immunomodulator to reduce lunglesions compared to negative control, thereby, providing evidence thatthis product has the potential to protect lung tissue during a BRDoutbreak. In this study, the day of treatment administration was notassociated with lung lesions thereby indicating that it did not matterif cattle received the DNA immunomodulator one day prior, or the sameday as, the onset of clinical signs associated with BRD. This outcome isimportant as the timing of exposure to BRD pathogens is generallyunknown among typical production systems and is further complicated bythe impact of various stressors experienced by cattle throughout thechain of production. Therefore, providing producers with a product thatoffers flexibility in the timing of administration, in relation to theonset of BRD, is of extreme value in the beef and dairy industries.

Example 3 Evaluation of Cattle Receiving a DNA Immunomodulator Two DaysBefore or Concurrently with an Experimental Challenge with Mannheimiahaemolytica

The purpose of this study was to determine the efficacy of the DNAimmunomodulator administered to calves two days before or concurrentlywith an experimental challenge with Mannheimia haemolytica.

Immunomodulator

The immunomodulator used in this study was the composition describedabove in Example 1.

Study Animals

96 Holstein steer calves weighing on average about 800-1000 lbs (363-454kg) were selected from a herd without a current history of respiratorydisease. Each individual calf was initially evaluated and determined tobe in good health. The 96 calves were divided into eight treatmentgroups of 12 calves each. Only animals not vaccinated for Mannheimiahaemolytica were included in the study. None of the animals had receivedan antimicrobial agent within 30 days prior to administration of DNAimmunomodulator. The treatment groups were administered varying doses ofthe DNA immunomodulator on the day of treatment as indicated in Table3.1 below. The dilution scheme of the DNA immunomodulator is provided inTable 3.2. The DNA immunomodulator was administered intramuscularly andcranial to the left shoulder, ventral to the nuchal ligament, andcaudo-dorsal to the jugular groove of the calves.

As referred to below, Treatment Day −2 refers to the start date of thestudy when Treatment Groups 1-3 were administered the immunomodulator.Treatment Day 0 is two days subsequent to Day −2, and so on.

TABLE 3.1 Administration Schedule of Immunomodulator and Mh ChallengeDNA Day of Day of Mh Animals Treat- Immuno- Immuno- Challenge per mentmodulator modulator Adminis- Treatment number Dose (μg) Administrationtration group 1 200 −2 0 12 2 50 −2 0 12 3 25 −2 0 12 4 200 0 0 12 5 500 0 12 6 25 0 0 12 7 0 (Control) −2 0 12 8 0 (Control) 0 0 12

Experimental Challenge

On Day 0, the calves were challenged with a total of 1.9×10¹⁰ CFUs. Theinoculum was administered via the respiratory tract.

Evaluation

As in the previous examples, on Days 1-5 of the study the calves wereevaluated for various health indicators. On Day 5, all calves wereeuthanized and necropsied. Lung lesion scores were determined for eachindividual calf at the time of necropsy.

FIG. 3.1 presents the model-adjusted lung lesion scores with respect todose of immunomodulator administered. FIG. 3.2 presents themodel-adjusted lung lesion scores with respect to day of immunomodulatoradministration.

Results

In this study, the dose of the DNA immunomodulator (i.e. 200 μg, 50 μg,and 25 μg) significantly reduced lung lesion scores compared to thenegative controls. However, no statistical differences in lung lesionscores were observed among the DNA immunomodulator dose groups. The dayof DNA immunomodulator administration (i.e. Days −2 and 0) wassignificantly associated with lung lesion scores. Significant reductionin lung lesions was observed when the immunomodulator was administeredon Day 0 when compared to Day −2.

Example 4 Mh Challenge Co-Administration of Immunomodulator and KilledMh Vaccine

The purpose of this study was to determine the efficacy of the DNAimmunomodulator co-administered with killed Mh vaccine to calvessubjected to an experimental challenge with Mannheimia haemolytica.

Immunomodulator

The immunomodulator used in this study was the composition describedabove in Example 1.

Study Animals

81 Holstein bull calves, 12 weeks old, were selected from a herd withouta current history of respiratory disease. Each individual calf wasevaluated and determined to be in good health. Only animals notvaccinated for Mannheimia haemolytica were included in the study. Noneof the animals had received an antimicrobial agent within 30 days priorto administration of inoculum.

Experimental Infection and Challenge

The challenge, or experimental infection, included exposure to aninoculum of Mannheimia haemolytica. The organisms were used at aconcentration of 1.7×10⁸ per animal for the first inoculum and 2.4×10¹⁰animal for the second inoculum. The animals were also challenged with aspray by another respiratory route. The concentration of the organismsin the spray inoculum was 1.9×10¹⁰ per animal.

The efficacy of the immunomodulator, as described above, administered tocalves followed by exposure to Mannheimia haemolytica was determined bythe twelve treatment groups as detailed on Table 3.

TABLE 4.3 Study Treatment Groups. Treatment Number Days of GroupTargeted Dose Day Contact Animals T1 Killed MH (oil) vaccine (SC) 0 X 7T2 Killed MH (oil) vaccine + 0 X 7 Immunomodulator 500 μg (SC) T3 KilledMH (oil) vaccine (SC) 7 X 6 T4 Killed MH (oil) vaccine + 7 X 7Immunomodulator 500 μg (SC) T5 Immunomodulator 500 μg (SC) 7 X 7 T6Immunomodulator 500 μg (SC) 13  X 7 T7 Immunomodulator 500 μg (IM) 13  X7 T8 Immunomodulator 500 μg (SC) 15* X 7 T9 Control NC NA NA 7 T10Control CC NA X 5 T11 Control SE NA X 7 T12 Killed MH (aqueous)vaccine + 0 X 7 Immunomodulator 500 μg (SC) Oil MH = Mannheimiahaemolytica vaccine (Pulmo-Guard ® PHM) Aqueous MH = Mannheimiahaemolytica vaccine (One Shot ®) NC = Not commingled and not spraychallenged (for background gross pathology) CC = Contact and spraychallenged SE = Used as Seeder challenge (Challenged intratracheal) Allanimals, except SE and NC were spray challenged SC = Subcutaneous routeof injection IM = Intramuscular route of injection NA = Not Applicable*Animals in group T8 will be treated after intranasal challenge

On day 0 of the study, all animals in groups T1, T2 and T12 wereadministered the immunomodulator subcutaneously. The immunomodulator wasadministered subcutaneously on Day 7 to Groups T3, T4, and T5. Theimmunomodulator was administered subcutaneously on Day 13 to Group T6and intramuscularly to T7. The immunomodulator was administeredsubcutaneously on Day 15 to Group T8.

All animals receiving the vaccine were vaccinated according to labelinstructions. Immunomodulator and the vaccine were administered as closetogether near a lymph node (neck)—two injections (one for vaccine andthe other for the immunomodulator). All animals receiving thesubcutaneous route of injection were injected near a lymph node in thesub scapular region.

On study day 10, all T11 calves were transported off site in a stocktrailer for approximately 24 hours to stress the calves. On Study day11, 20 mL of an inoculum containing Mannheimia haemolytica wasadministered transtracheally to all the T11 animals, followed 4 hourslater with 25 mL of inoculum. On study day 14, all groups, except T9were commingled and transported off site in a stock trailer forapproximately 24 hours to stress the calves. All animals except in groupNC were commingled in a large pen for 12 to 16 hours on Study day 14 andthen returned to their separate pens (each animal had a separate pen).On Study day 15, 20 mL of Mannheimia haemolytica was administered byanother respiratory route to all groups except T9 and T11. The animalswere observed daily throughout the study for clinical abnormalities andmortality. All animals were negative or had low titers at screeningprior to purchase of animals. The animals had high titers prior totreatment, which indicates that the animals serologically converted toMannheimia haemolytica prior to receiving treatment.

Results

The animals of group T8 had significantly lower lung lesions.

The study suggests that there is an onset of early protection (day 7)with or without vaccine (groups T4 and T5 compared to T3). See FIGS. 4.1and 4.2.

Example 5 Evaluation of Acquired Immunity in Cattle Vaccinated with aCommmercial-Live Vaccine When Co-Administered with a DNA Immunomodulator

The purpose of this study was to determine if co-administration of theDNA immunomodulator augmented the acquired immunity afforded bymodified-live viral (MLV) vaccines.

Immunomodulator

The immunomodulator used in this study was the composition describedabove in Example 1.

Study Animals

72 Holstein steers calves of weaning age were selected from a herdwithout a current history of respiratory disease. The 72 calves weredivided into six treatment groups of 12 calves each. Each individualcalf was evaluated and determined to be in good health. All calves werefree of serum antibodies to BHV-1, BVDV types 1 and 2, and BRSV. Inaddition, all calves were found to be serum antibody negative to PI-3.The calves were subsequently determined to be negative for bovine viraldiarrhea virus persistent infection by immunohistochemistry.

The treatment groups were administered the vaccine and varying doses ofthe DNA immunomodulator intramuscularly on the day of treatment asindicated in Table 5.1 below. The dilution scheme of the DNAimmunomodulator is provided in Table 5.2. On day 0 of the study, allanimals in groups T1-T4 were administered the immunomodulator. Allanimals receiving the vaccine were vaccinated according to labelinstructions. Immunomodulator and the vaccine were administered as closetogether cranial to the front of the shoulder—two injections (one forvaccine and the other for the immunomodulator).

TABLE 5.1 Administration Schedule of Immunomodulator and Vaccine Day ofVaccine and/or Number Immunomodulator of Group Targeted DoseAdministration Animals T1 MLV + Immunomodulator 0 12 (500 μg) IM T2MLV + Immunomodulator 0 12 (200 μg) IM T3 MLV + Immunomodulator 0 12(100 μg) IM T4 MLV + Immunomodulator 0 12 (50 μg) IM T5 MLV 0 12 T6 Notreatment NA 12 MLV = Mannheimia haemolytica vaccine (Bovi-shield ®) -modified-live 4-way viral respiratory vaccine IM = Intramuscular routeof injection

Evaluation

Immunological testing was performed on samples from appropriatehematological specimens collected from the calves on Days 0, 13, 28, 27,34 and 41. Cell mediated immunity (CMI) measurements were conducted foreach specimen. The target pathogens for this study were BHV-1, BVDV 1and 2, and BRSV. Laboratories used standardized procedures and methodsas appropriate for the previously specified target pathogens.

Results

Model-adjusted data for CMI outcomes on each Day of sample collectionamong all treatment groups were determined. Across all treatment groups,cell types, and antigens no statistical differences (P>0.10) weredetected when comparing DNA immunomodulator treatment groups—MLV vaccinecombinations to cattle receiving only the MLV vaccine (See FIGS.5.1-5.12). In particular, FIGS. 5.1-5.4 present the measurements of theCD 25 EI expression index (y-axis) across all five cell types for eachof the 6 treatment groups (x-axis). FIGS. 5.5-5.8 present themeasurements of the IFNγ expression index (y-axis) across all five celltypes for each of the 6 treatment groups (x-axis). FIGS. 5.9-5.12present the measurements of the IL-4 expression index (y-axis) acrossall five cell types for each of the 6 treatment groups (x-axis).Estimates were produced for each of the 4 BRD viral pathogensrepresented in their respective graph. For these statisticalevaluations, all comparisons were made to the “MLV only” treatmentgroup.

Statistically significant (P<0.10) treatment×Day interactions weredetected for BVDV 1 (Days 28 and 35) and BVDV 2 (Day 42). No significantfindings (P>0.10) were detected for BHV-1 at any of the listed timepoints. A graphical representation of these findings is displayed onFIGS. 5.13-5.15. The BRSV data was removed from analysis due toobservance of antibody seroconversion within the negative controltreatment group. Note that, for all statistical evaluations, allcomparisons were made to the “MLV only” treatment group.

Individual animal weights were also collected during the study. Agraphical representation of model-adjusted average daily gain outcomesis displayed in FIG. 5.16. No significant findings (P>0.10) weredetected across treatment groups when compared to the MLV only group.

In summary, the DNA immunomodulator did not enhance CMI whenco-administered with a MLV vaccine compared to the sole administrationof MLV vaccine. However, 500 μg of the DNA immunomodulator may augmenthumoral immunity when co-administered with a MLV vaccine (specificallyBVDV). Nonetheless, it should be noted that despite a lack of consistentimprovement in acquired immunity, co-administration of the DNAimmunomodulator, at doses of 500 μg, 200 μg, 100 μg, and 50 μg, did notimpair the positive immunologic effects induced by the MLV vaccine. Inaddition, performance (e.g. ADG) was not negatively impacted byadministration of the DNA immunomodulator.

Example 6 Evaluation of Acquired Immunity in Cattle Vaccinated with aCommmercial-Vaccine when Co-Administered with a DNA Immunomodulator

The purpose of this study was to determine if co-administration of theDNA immunomodulator augmented the acquired immunity afforded by vaccinescontaining inactivated antigens.

Immunomodulator

The immunomodulator used in this study was the composition describedabove in Example 1.

Study Animals

48 Holstein female cattle of 3-5 month age were selected from a herdwithout a current history of respiratory disease. The 48 cattle weredivided into six treatment groups of 8 animals each. Each individualanimal was evaluated and determined to be in good health. All animalswere free of serum antibodies to BHV-1, BVDV types 1 and 2. The animalswere also determined to be negative for bovine viral diarrhea viruspersistent infection by PCR. The animals were not selected on SNT titersagainst BRS virus and PI3 virus.

The treatment groups were administered the vaccine and varying doses ofthe DNA immunomodulator intramuscularly on the day of treatment asindicated in Table 5.1 below. The vaccine contained BHV1 and BVDV type1and 2 as inactivated antigens, and modified live PI3 virus and BRSvirus. The Immunomodulator and the vaccine were either given separatelyon the same side of the animal cranial to the front of the shoulder, orseparately on the opposite side of the animal in the some region, ormixed in one syringe. The dilution scheme of the DNA immunomodulator isprovided in Table 5.2.

TABLE 6.1 Administration Schedule of Immunomodulator and Vaccine Day ofVaccine and/or Number Immunomodulator of Group Targeted DoseAdministration Animals T1 Placebo (Dextrose 5%) 0 8 T2 Vaccine +Dextrose 0 8 IM, separately T3 Vaccine + Immunomodulator 0 8 (20 μg) IM,mixed T4 Vaccine + Immunomodulator 0 8 (200 μg) IM, mixed T5 Vaccine +Immunomodulator 0 8 (200 μg) IM, separately same side T6 Vaccine +Immunomodulator 0 8 (200 μg) IM, separately opposite side Vaccine =combined (inactivated and modified live)4-way viral respiratory vaccine(Rispoval ®) IM = Intramuscular route of injection

Evaluation

Immunological testing was performed on samples from appropriatehematological specimens collected from the cattle on Days 0, 3, 5, 7, 9,11, 14, 17, 20, 23 and 27. The target pathogens for this study wereBHV-1. BVDV 1 and 2. For information also the antibody titers againstBRS virus and PI3 virus were determined. Laboratories used standardizedSerum Neutralization Tests (SNT) as procedures for the previouslyspecified target pathogens.

Results

Statistically significant (P<0.010) treatment×Day interactions weredetected for BHV1 (Day 27). No significant findings (P>0.10) weredetected for all other time points for BHV1 and for BVDV type 1 at 2 atany of the listed time points. The results of the BRSV and PI3 titerswere not further evaluated because the animals were not serologicallynegative at the beginning of the study. An effect of treatment couldtherefore not be verified. A graphical representation of these findingsis displayed on FIG. 6.1. Note that, for all statistical evaluations,all comparisons were made to the “Vaccine and Dextrose5%” treatmentgroup.

What is claimed is:
 1. An immunomodulator composition, wherein theimmunomodulator composition comprises: a. a cationic liposome deliveryvehicle; and b. a nucleic acid molecule for treating bovine respiratorydisease in cattle.
 2. The composition of claim 1, wherein the liposomedelivery vehicle comprises lipids selected from the group consisting ofmultilamellar vesicle lipids and extruded lipids.
 3. The composition ofclaims 1 and 2, wherein the liposome delivery vehicle comprises pairs oflipids selected from the group consisting of DTMA and cholesterol; DOTAPand cholesterol; DOTIM and cholesterol, and DDAB and cholesterol.
 4. Thecomposition of claims 1 to 3, wherein the nucleic acid molecule is anisolated bacterially-derived nucleic acid vector without a gene insert,or a fragment thereof.
 5. The composition of claims 1 to 4, foradministration selected from the group consisting of intravenously,intramuscularly, intradermal, intraperitoneal, subcutaneously, byspray/aerosol, orally, intraocularly, intratracheally, and intranasal.6. The composition of claims 1 to 5, wherein the biological agent isselected from the group consisting of an immune enhancer proteins,immunogens, vaccines, antimicrobials or any combination thereof.
 7. Thecomposition of claim 1, wherein the bovine respiratory disease is causedby a viral infection and/or bacterial infection.
 8. The compositionaccording to any one of claims 1 to 7 for reducing clinical signs causedby Mannheimia haemolytica in cattle comprising an immunomodulatorcomposition, wherein the immunomodulator composition comprises: a. DOTIMand cholesterol lipid combination; and, b. nucleic acid molecule is anisolated bacterially-derived nucleic acid vector without a gene insert,or a fragment thereof.
 9. The composition of claim 8, further comprisinga biologic agent.
 10. The composition according to any one of claims 1to 9 for improving the acquired immune response of an animal that isadministered a vaccine, the composition comprises an immunomodulatorcomposition, wherein the immunomodulator composition comprises: a. DOTIMand cholesterol lipid combination; and, b. nucleic acid molecule is anisolated bacterially-derived nucleic acid vector without a gene insert,or a fragment thereof.
 11. The composition of claim 10, wherein theimmunomodulator composition is co-administered with the vaccine or isadministered after, before, or mixed with the vaccine.